Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules.

Novel fibronectin type II (Fn2)-module proteins were cloned from human and canine epididymal cDNA libraries. cDNA sequences predicted a highly conserved protein family, related but not homologous to ungulate seminal plasma proteins (approximately 50% sequence identity), and the first known examples of proteins with four tandemly arranged Fn2-domains. By Northern blot and in situ hybridization analyses the encoding mRNAs were shown to be abundant products of the epididymal duct epithelium, but not detectable in other tissues. Homologous mRNAs were identified in the epididymides of various mammals, representing members of this novel protein family of epididymal origin. Within the Fn2-module-encoding stretches, species homologues displayed >85% sequence identity, but showed high variability at their predicted N-termini. An antipeptide antiserum in Western blot analyses detected 30-35 kDa immunoreactive protein bands in epididymal tissue, cauda epididymidal fluid, and sperm membrane protein preparations. The tandem arrangement of increasing numbers of Fn2-modules might functionally correspond to the tendency to form oligomers that has been described for lipid-binding proteins.

Differential expression of novel abundant and highly regionalized mRNAs of the canine epididymis.

Three novel gene products have been cloned by differential screening of a dog epididymis cDNA library as part of a global appraisal of specific gene expression in the epididymis. The predicted proteins were provisionally named CE8-CE10 (for canine epididymal gene products 8-10). Northern blot analyses and in situ transcript hybridization confirmed that the cDNAs were all derived from tissue-specific, moderately to highly abundant mRNAs of the epididymal epithelium, showing a distinct regionalized expression pattern within the epididymal duct. Their sequences predict (i) a novel 19 kDa member of the Ly-6-domain protein superfamily (CE8), (ii) an approximately 30 kDa protein with multiple membrane-spanning regions (CE9), and (iii) a novel approximately 13 kDa single whey acidic protein domain protein (CE10). Closely related, cross-hybridizing gene products were abundant in the epididymis of stallions and bulls, but not in rodents or men. Changes in mRNA frequency were observed that specifically correlated with a cryptorchid situation and with the age of the dogs. Gene products restricted to the caput epididymidis were affected by both conditions, while those with a wider regional distribution were not.

Dog epididymis-specific mRNA encoding secretory glutathione peroxidase-like protein.

A differential library screening procedure was used to clone a novel abundant and tissue-specific cDNA from the dog epididymis. It was tentatively named CE7 for dog epididymal gene product 7. By sequence similarity to homologous counterparts expressed in mice, rats, pigs, and macaque monkeys, it appears that the 1.5 kb dog epididymal mRNA encodes the secretory glutathione peroxidase-like protein, GPX5. This protein is very similar to the family of glutathione peroxidase enzymes, but does not contain selenocysteine. Northern blot and in situ hybridization analyses revealed that the mRNA encoding CE7/GPX5, like its species homologues, was restricted to the epididymis and transcribed by the epithelial cells in the proximal parts of the organ. While the CE7 cDNA probe cross-hybridized to epididymal mRNAs in most species included in this study, it failed to identify a human GPX5 counterpart. Northern blot analyses of epididymal RNA extracts from hemi-cryptorchid dogs suggested that testicular secretions, including androgen hormones, temperature effects, or both, were involved in the region-dependent modulation of mRNA encoding CE7 in the dog epididymis. The effect was most obvious in the caput region of the abdominal organ where the mRNA encoding CE7 was almost completely downregulated.

The dog as a model system to study epididymal gene expression.

Advances in understanding the role of the epididymis in human sperm maturation and fertility are dependent upon the availability of appropriate model systems in which to examine regulatory mechanisms and functions in a controllable fashion. Of a number of mammalian species studied by us and others, we suggest that the dog epididymis offers an excellent compromise in terms of the similarity of the specific genes expressed to those in the human, their mode of regulation, and the availability of tissues suitable for cell culture studies. We have developed a set of important tools in the form of epididymis-specific canine cDNA clones and cell culture methods, with which to examine the functioning of the epididymal epithelial cells in vitro, as well as the expression of specific genes in vivo, under normal and pathological conditions (e.g. cryptorchidism). Using this model system, we have elaborated the regional patterns of gene expression in vivo for several of the major secretory protein genes of the canine epididymis, and we have examined the importance of androgens and temperature on gene expression in vitro.

Gene expression in the dog epididymis: a model for human epididymal function.

The physiology of the epididymis is an integral part of the maturation process by which human spermatozoa acquire the ability to reach and fertilize an oocyte. Because of the high degree of species specificity exhibited by the epididymal proteins involved in sperm maturation, we have assessed tissue from several alternative species for their suitability as a model for human epididymal physiology. Of these, the dog appears to offer an appropriate system. Northern hybridization using cDNA probes specific for human epididymal genes established that, irrespective of dog breed, the canine equivalents of the epididymis-specific HE1, HE4 and HE5 mRNAs were expressed highly in the canine epididymis. cDNA cloning and sequencing confirmed that the canine gene products, CE1, CE4 and CE5 were indeed true structural homologues of their human counterparts. Finally, tissue culture conditions were established wherein all three specific canine genes remained up-regulated after 5 days of culture. Thus, the prerequisite criteria for the development of a system which models human epididymal physiology are to a large degree fulfilled by this canine culture system.